Microvascular hyperpermeability represents an important injurious process underlying the development of many inflammatory diseases including diabetic complications. The long-term goal of our research program is to understand the cellular and molecular mechanisms in the regulation of microvascular barrier function under physiological and pathological conditions. As an integral component of the program, this project is designed to elucidate the signaling pathways and molecular mechanisms responsible for microvascular hyperpermeability during development of diabetes, a disease that affects a large population with high morbidity and mortality resulting from complications characterized by microvascular injury. Our central hypothesis states that diabetes upregulates PKCbeta in microvascular endothelium at multiple levels via MAPK-stimulated gene expression, PDK1-potentiated catalytic activity, and DAG-mediated kinase activation. We further propose that upregulated PKCbeta increases the paracellular permeability of venular endothelium by inducing endothelial cell contraction via the GDI-RhoA-ROCK cascade coupled with intercellular junction disorganization triggered by beta-catenin phosphorylation and VE-cadherin dissociation. Three specific aims are proposed: 1) to unequivocally establish the role of PKCbeta in microvascular leakage during diabetes; 2) to characterize the signaling mechanisms of PKCbeta upregulation in diabetic pigs; and 3) to elucidate the molecular basis of PKCbeta-elicited microvascular hyperpermeability. These aims will be accomplished through a multifaceted molecular physiology approach that incorporates molecular techniques with functional analyses at the microvascular level. A human-relevant pig model of diabetes will serve as the primary model for quantitative assessment of endothelial barrier function in intact microvessels. Data derived from this study will provide new insights into the pathogenesis of diabetic microvascular complications. Identification of the precise molecular mechanisms responsible for PKC-induced end-point injury may lead to a new avenue for searching therapeutic targets. Based on this study, a future direction of our research efforts will be directed to the development of molecular probes and therapies for diagnosis and treatment of microvascular leakage associated with chronic inflammatory diseases. [unreadable] [unreadable] [unreadable]